1. Field of the Invention
The present invention relates to novel methods, compositions, and elements for detecting nitrogen containing compounds capable of releasing ammonia upon enzymatic action.
2. Description of Related Art
There are several standard methods for the determination of urea. The Berthelot reaction utilizes the conversion of urea to ammonium carbonate by the action of urease followed by an oxidative condensation of ammonia with phenol catalyzed by sodium nitroprusside to form a blue indophenol. The condensation of diacetyl (or an oxime thereof) with urea in an acidic solution may be employed to produce a yellow diazine. In the Nesslerization procedure, urea is hydrolyzed by urease as in the Berthelot reaction and the ammonia produced is reacted with mercuric iodide and potassium iodide to yield (presumably) NH.sub.2 Hg.sub.2 I.sub.3 (red). A titrimetric procedure may be used in which urea is hydrolyzed by urease and the ammonia titrated with HCl to a Bromcresol green-methyl red endpoint. Urea concentration may also be determined potentiometrically by the immersion of an NH.sub.4.sup.+ sensitive electrode in a solution containing urea and urease. Finally the ammonia produced by the action of urease upon urea may be coupled with .alpha.-ketoglutaric acid in the presence of L-Glutamate:NAD oxoreductase (deaminating), E.C. 1.4.1.2 and nicotinamide adenine dinucleotide, reduced form, to yield L-glutamate and the oxidized form of the coenzyme. The reaction is followed at 340 nm.
The condensation reaction involving two equivalents of a .beta.-diketone and one each of ammonia and formaldehyde was first described by A. Hantzsch, Ann. Chem. Liebigs, 215, 1 (1882). Its only clinical usage has apparently been in the detection of glycerides in which liberated glycerol is oxidized to formaldehyde and condensed with 2,4-pentanedione and an ammonium salt to yield the tetra-substituted dihydropyridine. This procedure was reported by F. Dunsbach, Z. Klin. Chem., 4, 262 (1966) (N.B. CA 66:82993a). No suggestion is made to use this reaction to detect analyte other than glycerides.
The condensation reaction involving one equivalent of a .beta.-diketone and one of ammonia was described by E. Knoevenagel, Annalen, 281, 25 (1894). No suggestion is made of using this reaction to detect analyte other than ammonia.
U.S. Pat. No. Re 27,524 reissued Nov. 28, 1972 describes a process for measuring the amount of a nitrogen-containing compound in a sample, the enzyme being reactive with the nitrogen-containing compound which process comprises:
(1) mixing the sample with a compound that is enzymatically reactive with ammonia;
(2) adding to the nitrogen-containing compound an amount of enzyme sufficient to release ammonia at a measurable rate; and
(3) measuring the rate of reaction of ammonia with the compound that is enzymatically reactive with ammonia. The only compound reactive with ammonia which is specifically disclosed is .beta.-nicotinamide-adenine dinucleotide.
Creatinine has been detected as described in U.S. Pat. No. 3,806,416 issued Apr. 23, 1974 by converting creatinine to creatine using creatinine amidohydrolase and subsequently converting creatine to sarcosine and urea using creatine amidinohydrolase. As described below, this pair of reactions can be coupled to the instant urea assay to provide an assay for creatinine.
U.S. Pat. No. 3,992,158 issued Nov. 16, 1976 to Przybylowicz and Millikan describes unique integral elements for use in the qualitative and quantitative analysis of liquids such as blood serum and urine, which elements preferably comprise a porous spreading layer in fluid contact or communication with a reagent layer which comprises at least one material interactive with a component or decomposition product of a component of the liquid. This patent does not describe specifically compositions of the type described herein.
U.S. Pat. No. 3,145,086 describes a diagnostic composition for the determination of abnormally high blood urea, preferably impregnated on a cellulose strip, which composition combines urease with an indicator system including a buffer and an indicator material capable of changing color in the presence of a pH change. There is no suggestion of the use of urease in combination with a .beta.-diketone capable of condensing with ammonia and formaldehyde.
U.S. Pat. No. 3,873,269 describes a diagnostic composition for the determination of urea, preferably impregnated on an absorbent carrier such as filter paper, which combines urease with an indicator system capable of changing color in the presence of a pH change. There is no suggestion of the use of urease in combination with a diketone capable of condensing with ammonia.
Commonly-owned U.S. application Ser. No. 688,446 filed on May 20, 1976, U.S. Pat. No. 4,066,403, issued Jan. 3, 1978, entitled "Improved Multilayer Analytical Element" by B. Bruschi, describes a dry element having at least two reagents for generating a measurable indication of the presence of BUN, and a barrier composition separating the two reagents, the composition being selectively permeable to a decomposition product which will react with one of the reagents. Nowhere is there a specific disclosure that diketones should be one of the reagents, and the barrier layer would prevent the proper functioning of the present invention.